Enhancement of biological effects of oxidised nano- and microplastics in human professional phagocytes.

Micro and nanoplastics are ubiquitous pollutants that can cause adverse health effects even in humans. Effects of virgin and oxidised (simulating the aging processes) polystyrene nano (nPS) and micro particles (mPS) with diameters of 0.1 and 1 µm were studied on human professional phagocytes (i.e., monocyte cells THP-1 and macrophage-like mTHP-1 cells). After characterization by ATR-FTIR, UV-Vis spectroscopy, SEM and dynamic light-scattering analyses, the particles were FITC functionalised to quantify cellular uptake. Changes in the cell compartments were studied by acrydine orange and the pro-oxidant, cytotoxic and genotoxic effects were assessed. Phagocytosis was dose- and time- dependent and at 24 h 52% of nPS and 58% of mPS were engulfed. Despite the high homeostasis of professional phagocytes, significant ROS increases and DNA damage were observed after exposure to oxidised particles. The results highlight that the environmental aging processes enhances the adverse health effects of micro and nanoplastics.

Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity.

Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins.

C1q-Mediated Complement Activation and C3 Opsonization Trigger Recognition of Stealth Poly(2-methyl-2-oxazoline)-Coated Silica Nanoparticles by Human Phagocytes.

Poly(2-methyl-2-oxazoline) (PMOXA) is an alternative promising polymer to poly(ethylene glycol) (PEG) for design and engineering of macrophage-evading nanoparticles (NPs). Although PMOXA-engineered NPs have shown comparable pharmacokinetics and in vivo performance to PEGylated stealth NPs in the murine model, its interaction with elements of the human innate immune system has not been studied. From a translational angle, we studied the interaction of fully characterized PMOXA-coated vinyltriethoxysilane-derived organically modified silica NPs (PMOXA-coated NPs) of approximately 100 nm in diameter with human complement system, blood leukocytes, and macrophages and compared their performance with PEGylated and uncoated NP counterparts. Through detailed immunological and proteomic profiling, we show that PMOXA-coated NPs extensively trigger complement activation in human sera exclusively through the classical pathway. Complement activation is initiated by the sensing molecule C1q, where C1q binds with high affinity ( Kd = 11 ± 1 nM) to NP surfaces independent of immunoglobulin binding. C1q-mediated complement activation accelerates PMOXA opsonization with the third complement protein (C3) through the amplification loop of the alternative pathway. This promoted NP recognition by human blood leukocytes and monocyte-derived macrophages. The macrophage capture of PMOXA-coated NPs correlates with sera donor variability in complement activation and opsonization but not with other major corona proteins, including clusterin and a wide range of apolipoproteins. In contrast to these observations, PMOXA-coated NPs poorly activated the murine complement system and were marginally recognized by mouse macrophages. These studies provide important insights into compatibility of engineered NPs with elements of the human innate immune system for translational steps.

Modulating Phagocytic Cell Sequestration by Tailoring Nanoconstruct Softness.

The effect of nanoparticle size, shape, and surface properties on cellular uptake has been extensively investigated for its basic science and translational implications. Recently, softness is emerging as a design parameter for modulating the interaction of nanoparticles with cells and the biological microenvironment. Here, circular, quadrangular, and elliptical polymeric nanoconstructs of different sizes are realized with a Young's modulus ranging from ∼100 kPa (soft) to 10 MPa (rigid). The interaction of these nanoconstructs with professional phagocytic cells is assessed via confocal microscopy and flow cytometry analyses. Regardless of the size and shape, softer nanoconstructs evade cellular uptake up to 5 times more efficiently, by bone-marrow-derived monocytes, as compared to rigid nanoconstructs. Soft circular and quadrangular nanoconstructs are equally uptaken by professional phagocytic cells (<15%); soft elliptical particles are more avidly internalized (<60%) possibly because of the larger size and elongated shape, whereas over 70% of rigid nanoconstructs of any shape and size are uptaken. Inhibition of actin polymerization via cytochalasin D reduces the internalization propensity for all nanoconstruct types. High-resolution live cell microscopy documents that soft nanoconstructs mostly establish short-lived (<30 s) interactions with macrophages, thus diminishing the likelihood of recognition and internalization. The bending stiffness is identified as a discriminating factor for internalization, whereby particles with a bending stiffness slightly higher than cells would more efficiently oppose internalization as compared to stiffer or softer particles. These results confirm that softness is a key parameter in modulating the behavior of nanoparticles and are expected to inspire the design of more efficient nanoconstructs for drug delivery, biomedical imaging, and immunomodulatory therapies.

Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes.

Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (αMß2) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA toxin action, placing particular emphasis on signaling mechanisms by which the toxin-produced 3',5'-cyclic adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.

Molecular visualizing and quantifying immune-associated peroxynitrite fluxes in phagocytes and mouse inflammation model.

Reactions of peroxynitrite (ONOO-) with biomolecules can lead to cytotoxic and cytoprotective events. Due to the difficulty of directly and unambiguously measuring its levels, most of the beneficial effects associated with ONOO- in vivo remain controversial or poorly characterized. Recently, optical imaging has served as a powerful noninvasive approach to studying ONOO- in living systems. However, ratiometric probes for ONOO- are currently lacking. Herein, we report the design, synthesis, and biological evaluation of F482, a novel fluorescence indicator that relies on ONOO--induced diene oxidation. The remarkable sensitivity, selectivity, and photostability of F482 enabled us to visualize basal ONOO- in immune-stimulated phagocyte cells and quantify its generation in phagosomes by high-throughput flow cytometry analysis. With the aid of in vivo ONOO- imaging in a mouse inflammation model assisted by F482, we envision that F482 will find widespread applications in the study of the ONOO- biology associated with physiological and pathological processes in vitro and in vivo.

In vivo biodistribution and toxicity assessment of triplet-triplet annihilation-based upconversion nanocapsules.

Triplet-triplet annihilation (TTA)-based upconversion nanocapsules (UCNCs) have great potential in biological and medical applications. However, there are numerous unresolved issues with respect to the safety of these novel nanomaterials. In this work, for the first time, we studied the in vivo biodistribution of UCNCs which were synthesized by co-loading platinum (II)-tetraphenyl-tetrabenzoporphyrin (PtTPBP) and boron dipyrromethene derivative (BDP) into bovine serum albumin (BSA)-stabilized soybean oil droplets, and systematically assessed the potential toxicity of UCNCs both in vitro and in vivo. The results showed that UCNCs had no significant influence on the proliferation or the migration of HeLa cells even when the dosage was increased to 12 mg/mL. The biodistribution results demonstrated that UCNCs mainly accumulated in the mononuclear phagocyte system (MPS) including the liver and spleen after intravenous injection of the nanocapsules. When mice were intravenously injected with 1200 mg/kg of the UCNCs over a period of 60 days, no noticeable toxicity was observed under these treatment conditions as shown by body weight results, histological analyses, hematological analyses and blood biochemical examinations. This research inspires further studies on UCNCs for biomedical applications.

Clusterin in the protein corona plays a key role in the stealth effect of nanoparticles against phagocytes.

In biological fluids, nanoparticles interact with biological components such as proteins, and a layer called the "protein corona" forms around the nanoparticles. It is believed that the composition of the protein corona affects the cellular uptake and in vivo biodistribution of nanoparticles; however, the key proteins of the protein corona that control the biological fate of nanoparticles remain unclear. Recently, it was reported that clusterin binding to pegylated nanoparticles is important for the stealth effect of pegylated nanoparticles in phagocytes. However, the effect of clusterin on non-pegylated nanoparticles is unknown, although it is known that clusterin is present in the protein corona of non-pegylated nanoparticles. Here, we assessed the stealth effect of clusterin in the corona of non-pegylated silver nanoparticles and silica nanoparticles. We found that serum- and plasma-protein corona inhibited the cellular uptake of silver nanoparticles and silica nanoparticles in phagocytes and that the plasma-protein corona showed a greater stealth effect compared with the serum-protein corona. Clusterin was present in both the serum- and plasma-protein corona, but was present at a higher level in the plasma-protein corona than in the serum-protein corona. Clusterin binding to silver nanoparticles and silica nanoparticles suppressed the cellular uptake of nanoparticles in human macrophage-like cells (THP-1 cells). Although further studies are required to determine how clusterin suppresses non-specific cellular uptake in phagocytes, our data suggest that clusterin plays a key role in the stealth effect of not only pegylated nanoparticles but also non-pegylated nanoparticles.

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy.

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H(+) is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

[Modulating Effect of Extracellular HSP70 on Generation of Reactive Oxigen Species in Populations of Phagocytes].

Reactive oxygen species (ROS) produced by phagocytic cells of the innate immune system play an important role in the first line of defense protecting the host from pathogens. The NADPH oxidase multi-subunit complex is the main source of ROS in all types of the phagocytes. Formation of the membrane-associated enzyme complex and its activity are dependent on many different factors controlling both intensification and suppression of the ROS production rate. However, the evidences are emerging in recent years indicating existence of poorly studied mechanisms of restriction of ROS generation level in phagocytes directed at protection of host tissues in the sites of inflammation from destruction caused by the oxygen free radicals. Our previous data and results of other authors demonstrate that a mechanism of the limitation of ROS production by phagocytes may by connected with immunomodulating activity of extracellular pool. of HSP70. In the present work, we used inhibitors of NADPH oxidase and in vitro cultures of different phagocytes to study a possible relationship between down-regulating effect of exogenous HSP70 on ROS generation and the interaction of the protein with the enzyme subunits. Our results confirmed the literature data concerning the ability of extracellular HSP70 to modulate NADPH oxidase activity and demonstrated for the first time an inhibitory effect of the protein on intracellular ROS generation in phagocytes.

Improvement of a rapid screening test for chronic granulomatous disease.

Diagnosis of CGD is made by demonstrating absent or markedly reduced oxidase activity in stimulated neutrophils. The screening test proposed is based upon the naked eye evaluation of the reduction of NBT on a solid surface. It seems to be a useful tool for rapid and inexpensive detection of CGD patients, especially for large-scale screening purposes. The test was carried out on forty-five subjects: two males affected by CGD, three female carriers and forty healthy donors. The test confirmed the results obtained with flow cytometric and NBT assays.

Induction of mouse melioidosis with meningitis by CD11b+ phagocytic cells harboring intracellular B. pseudomallei as a Trojan horse.

BACKGROUND: Approximately 3-5% of patients with melioidosis manifest CNS symptoms; however, the clinical data regarding neurological melioidosis are limited. METHODS AND FINDINGS: We established a mouse model of melioidosis with meningitis characterized by neutrophil infiltration into the meninges histologically and B. pseudomallei in the cerebrospinal fluid (CSF) by bacteriological culturing methods. As the disease progresses, the bacteria successively colonize the spleen, liver, bone marrow (BM) and brain and invade splenic and BM cells by days 2 and 6 post-infection, respectively. The predominant cell types intracellularly infected with B. pseudomallei were splenic and BM CD11b(+) populations. The CD11b(+)Ly6C(high) inflamed monocytes, CD11b(+)Ly6C(low) resident monocytes, CD11b(+)Ly6G(+) neutrophils, CD11b(+)F4/80(+) macrophages and CD11b(+)CD19(+) B cells were expanded in the spleen and BM during the progression of melioidosis. After adoptive transfer of CD11b populations harboring B. pseudomallei, the infected CD11b(+) cells induced bacterial colonization in the brain, whereas CD11b(-) cells only partially induced colonization; extracellular (free) B. pseudomallei were unable to colonize the brain. CD62L (selectin) was absent on splenic CD11b(+) cells on day 4 but was expressed on day 10 post-infection. Adoptive transfer of CD11b(+) cells expressing CD62L (harvested on day 10 post-infection) resulted in meningitis in the recipients, but transfer of CD11b(+) CD62L-negative cells did not. CONCLUSIONS/SIGNIFICANCE: We suggest that B. pseudomallei-infected CD11b(+) selectin-expressing cells act as a Trojan horse and are able to transmigrate across endothelial cells, resulting in melioidosis with meningitis.

Noninvasive molecular imaging of tuberculosis-associated inflammation with radioiodinated DPA-713.

BACKGROUND: Increased expression of translocator protein (TSPO) is a feature of microglial and macrophage activation. Since activated macrophages are key components of tuberculosis-associated inflammation, we evaluated radioiodinated DPA-713, a synthetic ligand of TSPO, for in vivo imaging of host response. METHODS: Mice were infected with aerosolized Mycobacterium tuberculosis and evaluated using whole-body [(125)I]iodo-DPA-713 single-photon emission computed tomography (SPECT). Ex vivo biodistribution and correlative immunofluorescence studies were also performed. RESULTS: [(125)I]Iodo-DPA-713 SPECT imaging clearly delineated tuberculosis-associated pulmonary inflammation in live animals. Biodistribution studies confirmed radiotracer specificity for inflamed pulmonary tissues. Immunofluorescence studies demonstrated that TSPO is highly expressed in CD68(+) macrophages and phagocytic cells within tuberculosis lesions and that [(125)I]DPA-713 specifically accumulates within these cells. Coadministration of excess unlabelled DPA-713 abrogated both the SPECT and ex vivo fluorescence signals. Lesion-specific signal-to-noise ratios were significantly higher with [(125)I]iodo-DPA-713 SPECT (4.06 ± 0.52) versus [(18)F]fluorodeoxyglucose (FDG) positron emission tomography (PET) (2.00 ± 0.28) performed in the same mice (P = .004). CONCLUSIONS: [(125)I]Iodo-DPA-713 accumulates specifically in tuberculosis-associated inflammatory lesions by selective retention within macrophages and phagocytic cells. [(125)I]Iodo-DPA-713 SPECT provides higher lesion-specific signal-to-noise ratios than [(18)F]FDG PET and may prove to be a more specific biomarker to monitor tuberculosis in situ.

S100A8 and S100A9-oxidant scavengers in inflammation.

S100A8 and S100A9 are generally considered proinflammatory. Hypohalous acids generated by activated phagocytes promote novel modifications in murine S100A8 but modifications to human S100A8 are undefined and there is no evidence that these proteins scavenge oxidants in human disease. Recombinant S100A8 was exquisitely sensitive to equimolar ratios of HOCl, which generated sulfinic and sulfonic acid intermediates and novel oxathiazolidine oxide/dioxide forms (mass additions, m/z +30 and +46) on the single Cys42 residue. Met78(O) and Trp54(+16) were also present. HOBr generated sulfonic acid intermediates and oxidized Trp54(+16). Evidence for oxidation of the single Cys3 residue in recS100A9 HOCl was weak; Met63, Met81, Met83, and Met94 were converted to Met(O) in vitro. Oxidized S100A8 was prominent in lungs from patients with asthma and significantly elevated in sputum compared to controls, whereas S100A8 and S100A9 were not significantly increased. Oxidized monomeric S100A8 was the major component in asthmatic sputum, and modifications, including the oxathiazolidine adducts, were similar to those generated by HOCl in vitro. Oxidized Met63, Met81, and Met94 were variously present in S100A9 from asthmatic sputum. Results have broad implications for conditions under which hypohalous acid oxidants are generated by activated phagocytes. Identification in human disease of the novel S100A8 Cys derivatives typical of those generated in vitro strongly supports the notion that S100A8 contributes to antioxidant defense during oxidative stress.

Phagocytes mediate targeting of iron oxide nanoparticles to tumors for cancer therapy.

Nanotechnology has great potential to produce novel therapeutic strategies that target malignant cells through the ability of nanoparticles to get access to and be ingested by living cells. However its specificity for accumulation in tumors, which is the key factor that determines its efficacy, has always been a challenge. Here we tested a novel strategy to target and treat ovarian cancer, a representative peritoneal cancer, using iron oxide nanoparticles (IONPs) and an alternating magnetic field (AMF). Peritoneal tumors in general are directly accessible to nanoparticles administered intraperitoneally (IP), as opposed to the more commonly attempted intravenous (IV) administration. In addition, tumor-associated immunosuppressive phagocytes, a predominant cell population in the tumor microenvironment of almost all solid tumors, and cells that are critical for tumor progression, are constantly recruited to the tumor, and therefore could possibly function to bring nanoparticles to tumors. Here we demonstrate that tumor-associated peritoneal phagocytes ingest and carry IONPs specifically to tumors and that these specifically delivered nanoparticles can damage tumor cells after IONP-mediated hyperthermia generated by AMF. This illustrates therapeutic possibilities of intraperitoneal (IP) injection of nanoparticles and subsequent ingestion by tumor-associated phagocytes, to directly impact tumors or stimulate antitumor immune responses. This approach could use IONPs combined with AMF as done here, or other nanoparticles with cytotoxic potential. Overall, the data presented here support IP injection of nanoparticles to utilize peritoneal phagocytes as a delivery vehicle in association with IONP-mediated hyperthermia as therapeutic strategies for ovarian and other peritoneal cancers.

Gender differences in the immune system activities of sea urchin Paracentrotus lividus.

In the immune system of vertebrates, gender-specific differences in individual immune competence are well known. In general, females possess more powerful immune response than males. In invertebrates, the situation is much less clear. For this purpose we have chosen to study the immune response of the two sexes of the echinoderm Paracentrotus lividus in pre- and post-spawning phases. The coelomic fluid from the echinoderms contains several coelomocyte types and molecules involved in innate immune defenses. In this article we report that the degree of immune responses in the P. lividus differs according to sex in both pre- and post-spawning phases. We found in all tests that females were more active than males. The results indicate that females possess a significant higher number of immunocytes consisting of phagocytes and uncolored spherulocytes. Since the immunological activity is mainly based on immunocytes, it was not surprising that females possessed the highest values of cytotoxicity and hemolysis activity and showed a greater ability to uptake neutral red and phagocyte yeasts cells, while the average number of ingested particles per active phagocyte was not significantly different. Furthermore, agglutinating activity was more evident in the coelomocyte lysate and coelomic fluid of females than in those of males. Finally we found that the acidic extract of female gonads possessed greater antimicrobial activity than that of male gonads. These results make it very likely that gender differences in the immune response are not restricted to vertebrates; rather, they are a general evolutionary phenomenon.

A rapid flow cytometric method for distinguishing between febrile bacterial and viral infections.

Antibiotic resistance due to the inappropriate use of antimicrobials is one of the most critical public health problems worldwide. A major factor underlying the unnecessary use of antibiotics is the lack of rapid and accurate diagnostic tests. Therefore, we aimed to develop a novel rapid flow cytometric method for distinguishing between febrile bacterial and viral infections. In this prospective comparative study, quantitative flow cytometric analysis of FcγRII/CD32, CR1/CD35, MHC Class I receptor (MHCI), and C5aR/CD88 on human phagocytes was performed in 286 hospitalized febrile patients with suspected infection. After using microbiological and serological detection methods, or clinical diagnosis, 205 patients were identified with either bacterial (n=136) or viral (n=69) infection. Receptor data from patients were compared to those of 50 healthy controls. We developed a flow cytometric marker of local and systemic bacterial infections designated "bacterial infection score (BIS)" incorporating the quantitative analysis of FcγRII/CD32, CR1/CD35, C5aR/CD88 and MHCI on neutrophils and/or monocytes, which displays 91% sensitivity and 92% specificity in distinguishing between microbiologically confirmed bacterial (n=77) and serologically confirmed viral infections (n=61) within 1h. The BIS method was effectively applied to distinguish between bacterial and viral (pandemic H1N1 influenza) pneumonia cases with 96% sensitivity and 92% specificity. We propose that the rapid BIS test can assist physicians in deciding whether antibiotic treatment is necessary, thus reducing unnecessary antimicrobial use.

Exploiting 4-sulphate N-acetyl galactosamine decorated gelatin nanoparticles for effective targeting to professional phagocytes in vitro and in vivo.

The present study was focused on the development of surface modified gelatin nanoparticles (SGNPs) using novel ligand 4-sulfated N-acetyl galactosamine (4-SO(4)GalNAc) for specific targeting to macrophages. The gelatin has been modified with the potential targeting moiety 4-SO(4)GalNAc, which was further used for the preparation of modified nanoparticles. The nanoparticles have been prepared by two step desolvation method. The SGNPs and unmodified gelatin nanoparticles (GNPs) were loaded with doxorubicin (DxR) and its targeting potential was compared. Developed DxR-loaded SGNPs (DxR-SGNPs) were found to have negative zeta potential (-19.8 ± 0.22 mV) whereas DxR-loaded GNPs (DxR-GNPs) have the positive zeta potential of around +12.2 ± 0.36 mV. The mean particle size of DxR-SGNPs and DxR-GNPs was found to be 283 ± 7 and 134 ± 5 nm, respectively. Flow cytometric data confirmed the enhanced uptake of DxR-SGNPs in J774A.1 and PBMC when compared with DxR-GNPs. Intracellular localization studies indicate that the fluorescence intensity of DxR-SGNPs was significantly higher when compared to DxR-GNPs. DxR-SGNPs rendered significantly higher localization of DxR in liver and spleen as compared to DxR-GNPs after i.v. administration. The study stipulates that 4-SO(4)GalNAc assures for targeting resident macrophages.

Purification of pathogen vacuoles from Legionella-infected phagocytes.

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"(1). In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles(2-4). The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics. The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania(5), Listeria(6), Mycobacterium(7), Rhodococcus(8), Salmonella(9) or Legionella spp.(10). However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment. The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)(3,11) . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation(12,13) (Fig. 1). A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before(13). LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.